Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators.

TitleIdentification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators.
Publication TypeJournal Article
Year of Publication2016
AuthorsMathias, RA, Greco, TM, Cristea, IM
JournalMethods Mol Biol
Volume1436
Pagination213-39
Date Published2016
ISSN1940-6029
Abstract

Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates. Specifically, SIRT4 was found to delipoylate and modulate the activity of the pyruvate dehydrogenase complex (PDH), a protein complex critical for the production of acetyl-CoA. Furthermore, SIRT4 is well known to have ADP-ribosyltransferase activity and to regulate the activity of the glutamate dehydrogenase complex (GDH). Adding to its impressive range of enzymatic activities are its ability to deacetylate malonyl-CoA decarboxylase (MCD) to regulate lipid catabolism, and its newly recognized ability to remove biotinyl groups from substrates that remain to be defined. Given the wide range of enzymatic activities and the still limited knowledge of its substrates, further studies are needed to characterize its protein interactions and its impact on metabolic pathways. Here, we present several proven protocols for identifying SIRT4 protein interaction networks within the mitochondria. Specifically, we describe methods for generating human cell lines expressing SIRT4, purifying mitochondria from crude organelles, and effectively capturing SIRT4 with its interactions and substrates.

DOI10.1007/978-1-4939-3667-0_15
Alternate JournalMethods Mol. Biol.
PubMed ID27246218
PubMed Central IDPMC4919573
Grant ListR01 GM114141 / GM / NIGMS NIH HHS / United States
R01 HL127640 / HL / NHLBI NIH HHS / United States
R21 AI102187 / AI / NIAID NIH HHS / United States